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. 2013 May 15;24(10):1559–1573. doi: 10.1091/mbc.E12-12-0850

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© 2013 Mitchison et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

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FIGURE 8: — Aurora B and Kif4 are required for plus-end alignment. (A) Aurora B and Kif4 depletion. HSS was depleted with anti-Aurora B, anti-Kif4, or control IgG and analyzed by immunoblotting. Depletion was >90% in both cases. In some experiments Kif4-GFP was added back to approximately endogenous levels. (B) Aurora B (red)–depleted compared with control IgG-depleted pineapples at 60 min (40× confocal). Note that plus-end alignment is lost after Aurora B depletion, and the antibody probe to Aurora B (red) did not localize. NUMA (blue) localization was not affected by Aurora B depletion. (C) Aurora B inhibition. Aurora B activity was inhibited with 4 μM hesperadin in this experiment. At 20 min hesperadin-treated reactions were poorly organized compared with control. At 60 min minus ends always aligned and recruited NUMA. Plus ends aligned in some assemblies as shown, although others resembled Aurora B (red)–depleted assemblies. Hesperadin inhibited, but did not completely block, recruitment of the Aurora B probe to microtubules (compare black and white panels in B and C, which were scaled the same). Plus-end accumulation of the probe was strongly inhibited. Similar results were obtained at 1.4 and 16 μM Hesperadin and with other small-molecule inhibitors (see text). (C′) Control reaction for C run in parallel with no drug. (D) Kif4 depletion. HSS was depleted with anti-Kif4 or control IgG, and pineapple assembly was run in parallel (20×, wide field, tubulin probe only). Depletion of Kif4 (bottom) had no effect on initial aggregation or minus-end alignment. Plus ends in the center appeared to grow longer than control and failed to align, resulting in assemblies that did not hollow out. Microtubules accumulated in whorls in the center in Kif4-depleted pineapples (35 min, 4× panel), suggesting disregulated plus-end growth. (E) Kif4-GFP addback (40× confocal, 100-min time point). Kif4 depletion (left) blocked plus-end focusing and alignment as in B. Aurora B was still recruited to microtubules but did not accumulate in obvious foci, probably because plus ends were distributed. Addback of Kif4-GFP (middle) rescued plus-end alignment and Aurora B accumulation and accumulated at plus ends as in A. Depletion with random IgG (right) had no effect.